Neoplastic transformation produces many changes in cell physiology. Growth-promoting hormones produce some of these same changes. Endocytosis is a process that regulates both the interaction of cells with growth-promoting hormones and the entry of transforming viruses. In the last few years, our study of the pathway of endocytosis in cultured cells has revealed that epidermal growth factor (a growth promoting hormone)(EGF) and transferrin (a plasma iron-binding protein necessary for cell growth)(TF) are co-internalized in the same pathway into human carcinoma cells, but diverge from each other in the trans-reticular network of the Golgi system. Cytochemical experiments using electron microscopy have shown that the receptors for EGF and TF are also internalized with the ligands. However, EGF and its receptor have been found to be delivered to lysosomes and degraded, whereas TF and its receptor are recycled intact back to the cell surface. The morphologic divergence of these two ligand-receptor types appears to involve clathrin-coated pits of the Golgi system. We have now shown that cell fractionation using an anti-clathrin affinity absorbant technique revealed the same separation of TF and EGF seen morphologically. It was also shown that the entry of the EGF receptor could be at least partially induced by treatment of cells with phorbol esters in the absence of EGF. To evaluate the site of ligand-receptor sorting, we employed new markers for the Golgi and lysosomal systems, including: (1) Limax flavus lectin labeling of sialic acid residues in the trans-Golgi system, (2) LAMP-1 monoclonal antibody to a lysosomal membrane protein, (3) ABL-70 monoclonal antibody to a Golgi stack marker protein, (4) antibody to MEP as a marker of lysosomal matrix proteins, and (5) 2C6 monoclonal antibody as a marker for the alpha2-macroglobulin receptor. We examined the morphologic kinetics of delivery of ligand into the lysosomal system in double-label experiments using LAMP-1 as a marker for lysosomal membranes. We also devised a new method for preservation of membrane proteins for immunofluorescence detection.